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Revealing Ligand & GPCR Bias Using A Combination of 2 nd Messenger, β-Arrestin & Internalization EFC Assay Formats

GPCR Ligand Bias Assays

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DiscoveRx GPCR cell based assays for identifying novel ligands, studying receptor pharmacology and uncovering ligand bias.

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Page 1: GPCR Ligand Bias Assays

Revealing Ligand & GPCR Bias Using A Combination

of 2nd Messenger, β-Arrestin & Internalization

EFC Assay Formats

Page 2: GPCR Ligand Bias Assays

DiscoveRx – Your Drug Discovery Partner

Catalog Products

Profiling and Screening

Custom Assay

Development

Cell-Based Assays246 GPCR cell lines

15 Cell Based Kinases26 NHR cell lines6 Pathway assays

Page 3: GPCR Ligand Bias Assays

DiscoveRx Core Technology

Cell Biology

• Cellular GPCR Assays• Cellular NHR Assays• Cellular Kinase Assays • Cellular Protease Assays• Pathway / Signaling Assays

PathHunterTM Product Line

• GPCR Signaling Assays• Generic Kinase Assays• Protease Technology• Unactive Kinase Technology

HitHunterTM Product Line

Biochemical

+

Inactive Fragments Active Enzyme

PK PK

Active EnzymeEnzyme

Complementation

Page 4: GPCR Ligand Bias Assays

Evolution of Understanding GPCR Function

1.Binding - On/ Off Radioligand binding studies

2. Receptor Activation Through Ga Second messenger assays

3. Receptor De-sensitization via b-ArrestinInternalization & recruitment studies

Page 5: GPCR Ligand Bias Assays

Evolution of Understanding GPCR Function4. b-Arrestin involved in desensitization but also has signaling properties

Kinases (MAPK, P3K, AKT)

5. Novel conformations of the same receptor can initiate different signaling cascades Now understood that agonists can activate only a subset of signals

Balanced Signal

Biased Signals

Originally thought that all downstream pathways equally activated for a given agonist

Page 6: GPCR Ligand Bias Assays

Evolution of GPCRs

Page 7: GPCR Ligand Bias Assays

Functional GPCR Activities & Ligand Bias

Arrestin

Second MessengerSignaling

cAMP

Calcium

Internalization

Events following ligand binding to GPCRs

Differential activation of these pathways occurs using the

same ligand (Ligand Bias)

Page 8: GPCR Ligand Bias Assays

GPCR Activation: Multiple Biological Processes

cAMPCalciu

m

G-protein Activation

Arrestin Recruitment

Internalization

GRK

• 2nd Messenger Signaling

• Alternate pathway• Short term

desensitization• Contributes to

Internalization

• Long term desensitization

• Altered Trafficking• Degradation /

Recycling

Opportunity for ligand

bias

1

Long Acting Agonists

2

Avoid Unwanted Signaling

3

Functional Antagonists, Receptor removal

Monitoring each function can aid in compound characterization

Page 9: GPCR Ligand Bias Assays

PathHunter™ Protein Interaction Technology

+

Inactive Fragments Active Enzyme

PKPK

Active Enzyme

Weak/No activity High activity

EnzymeComplementation

+

Modified EnzymeComplementation

for Protein interactions

Features:• Generic, homogeneous approach applicable to a wide range of drug target classes• Flexible, small peptide tag with options for high and low affinity• Only technology successfully transitioned from biochemical to cell based format

Page 10: GPCR Ligand Bias Assays

GPCR Activation & b-Arrestin Binding

Small protein tag (42 aa) Dynamic protein interaction assay Target-specific signal Chemiluminescent or fluorescent

detection Transfers easily from benchtop to

full HTS campaigns10-11 10-10 10-9 10-8 10-7 10-6 10-50

10000

20000

30000

40000

50000

Isoproterenol [M]

RL

U

PathHunter™ b-Arrestin Assays

ADRB2

Page 11: GPCR Ligand Bias Assays

Arrestin-Based GPCR Signaling

10-1410-1310 -1210-1110-10 10-9 10-8 10-7 10-6 10-50

10000

20000

30000

40000

50000

Isoproterenol [M]

RL

U

SSTR2 (Gi) CHRM5 (Gq)

ADRB2 (Gs)

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -60

500

1000

1500

2000

2500

3000

3500

Somatostatin 28

RL

U

EC50=2.0 nM S/B=42 ACHR5

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30

2000

4000

6000

8000

10000

12000

14000

Oxotremorine M

RL

U

EC50=3.1 μM; S/B=5.9

EC50=11.2 nM S/B=6.6

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

10000

20000

30000

40000

Glucagon [M]R

LU

EC50=15.3nM; S/B=11

Type B Glucagon (GCGR)

1 hr1 hr

Read Luminescence

1 hour

1 hour

Thaw & Plate Cells

12-48 hrs

Page 12: GPCR Ligand Bias Assays

Highlights of PathHunter™ Arrestin Format Stochiometric Signal

GenerationCompound EfficacyAntagonist Mode

Predictable Pharmacology

Specificity Serum samples

• Chemokines • Bioactive lipids • Antibody Supernatants

CHO A2 ADRB2 Clone Ligand Rank Order

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30

250

500

750

1000

1250

1500

1750SalbutamolIsoproterenol(-) EpinephrineDeoxyepinephrineClenbuterolSalmeterolDobutamineFormoterolIsoetharineMetaproterenolRitodrineTerbutalineAgonist [M]

RL

U

CHO A2 GLP2R

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50

2500

5000

7500

10000

GLP 2 (1-33) human [M]

RL

U

10-8 10-7 10-6 10-5 10-4 10-30

1000

2000

3000

4000

5000

6000

7000

8000

20% FBS40% FBS60% FBS80% FBS

Anti-CCL20 mAb [g/mL] + 10nM CCL20

RL

U

Page 13: GPCR Ligand Bias Assays

PathHunter Performance

Real-time analysis

Signal to noise

FormoterolClenbuterol

Isoproterenol

ADRB2

PathHunter clones (Receptors tested with

reference agonist)

S:B

Rati

o

>50% are 9X or better

Page 14: GPCR Ligand Bias Assays

Arrestin is G-protein Independent

G-protein inhibitor independent

CHO Arrestin FPRL1

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50

10000

20000

30000

40000

50000

60000(+) Pertussis tox(-) Pertussis tox

WKYMVm-NH2 [M]

RL

U

CHO Gi FPRL1

10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60

250

500

750

1000

1250

(+) Pertussis tox(-) Pertussis tox

WKYMVm-NH2 [M] + 20 M Forskolin

RL

U

cAMP Assay Arrestin Assay

Arrestin binding and internalization can occur in the absence of G-protein activity

CXCR7

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

10000

20000

30000

Complement C5a [M]

RL

Us

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60

10000

20000

30000

40000

SDF1a [M]

RL

U

C5L2

Lambertus et al. J. Biomol Screening (2009)

Arrestin in the absence of G-protein activation

Basis for Arrestin Ligand Bias

Page 15: GPCR Ligand Bias Assays

Second Messenger

Native Gi, Gs, and Gq coupled cell lines HitHunter® biochemical reagents for cAMP detection Complex pharmacology- Agonists, antagonists, partial agonists & allosterism Differentiating compounds - Large assay windows, ideal for difficult Gi targets Miniaturizable Fluorescent and Aequorin-based calcium detection

Page 16: GPCR Ligand Bias Assays

10-13 10-12 10-11 10-10 10-9 10-8 10-70

1000

2000

3000

4000

Angiotensin II [M]

RL

U

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

1000

2000

3000

4000

[Sar1, Ile4,8]-Angiotensin II [M]

RL

U

10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-80

250

500

750

1000

1250

1500

Angiotensin II [M]

RL

U

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

250

500

750

1000

1250

1500

[Sar1, Ile4,8]-Angiotensin II [M]

RL

U

Arrestin Biased Agonism

Biased agonist SII

Reference agonist

Biased agonist SII

Reference agonist

Ca++

AGTR1

Page 17: GPCR Ligand Bias Assays

10-1310-1210 -1110-10 10-9 10-8 10-7 10-6 10-5 10-40

500

1000

1500

2000

Ligand [M]

RL

U

GlucagonDHG

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

250

500

750

1000

1250

Ligand [M]

RL

U

Glucagon

DHG+ 300nM Glucagon

cAMP

GCGR

DHG is a ligand for the GCGR receptor-- antagonist in Arrestin but a partial agonist in

cAMP

PathHunter is Ideal for Investigating Ligand Bias

Page 18: GPCR Ligand Bias Assays

Lessons Learned

7TMR SignalingBoth G-protein dependent & independent

pathwaysLinked but not dependent pathwaysCan be modulated independently by

compounds

PathHunter™ Arrestin TechnologyBroadly applicable measure of arrestin

signalingIdeal for difficult targets and orphansHTS friendlyOpportunity for multiplexed measurements

Page 19: GPCR Ligand Bias Assays

GPCR Internalization

Applicable to the majority of GPCRs

Reduces the number of cell surface receptors acutelyRecycledTargeted for degradationFunctional antagonism

Compartmentalized signaling has been observed

Page 20: GPCR Ligand Bias Assays

EAEA

EA

Complemented Enzyme

Arr

esti

n R

ecru

itm

en

t

- Gain-of-signal, large S:B- Arrestin dependent-Short-term desensitization at membrane

PathHunter™ GPCR Internalization Assays:2 Convenient Platforms. Same Answer. Does the receptor internalize?

Endo

EAEA

EA

Complemented EnzymeA

cti

vate

d I

nte

rnaliza

tion

- Gain-of-signal, large S:B- Arrestin dependent- Long-term desensitization at endosome

EA

EA

Page 21: GPCR Ligand Bias Assays

10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60

250

500

750

1000

CCL3 [M]

RL

U

10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30

250

500

750

Oxotremorine-M [M]

RL

U

CCR5 M5NTSR1

Neurotensin Chemokine Muscarinic

PathHunter™ Activated GPCR Internalization Assays:Broadly Applicable

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-50

2500

5000

7500

S/B = 47.0

BOTTOM TOP LOGEC50 HILLSLOPE EC50

NTSR1177.46785-8.8501.6511.4128e-009

Neurotensin [M]

RL

U

Tested across 35 different families Over 65 targets available (over 80 by

3/1) Building out specific panels

Compound selectivity Timing, magnitude, cell type

specificity

Page 22: GPCR Ligand Bias Assays

GPCR “Trio Project” For Ligand Bias

24 targets tested in all 3 formats, average of 5 agonists per target

Pharmacology differences observed in 11 of 24 Expected overlap between cAMP/Calcium and ArrestinOnly 1% of ligands are completely biased, 5-10%

show some biasMore differences with internalization

Currently expanding studies and following up on “biased” ligands

22

Page 23: GPCR Ligand Bias Assays

cAMP

0.00

010.

001

0.01 0.

1 1 10 100

1000

0

25

50

75

100

125CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8

Agonist + 15 M Forskolin

% c

AM

P I

nh

ibit

ion

Internalization

0.00

010.

001

0.01 0.

1 1 10 100

1000

0

25

50

75

100

125

150CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8

Agonist

% A

ctiv

atio

n

Arrestin

-4.0

0-3

.00

-2.0

0-1

.00

0.00

1.00

2.00

3.00

0

25

50

75

100

125

150CXCL1CXCL2CXCL3CXCL5CXCL6Interleukin-8

Agonist

% A

ctiv

atio

n

Arr

estin

No Bias detectedCXCR2

Inte

rnal

izat

ion

2nd M

esse

nger

DiscoveRx Trio Initiative

Arrestin

0.000

10.0

01 0.01 0.1 1 10 10

010

00

1000

0

0

25

50

75

100

125

150CCL3

CCL23

Agonist

% A

ctiv

atio

n

cAMP

0.000

10.0

01 0.01 0.1 1 10 10

010

00

1000

0

0

25

50

75

100

125

150CCL3CCL23

Agonist + 15 M Forskolin

% c

AM

P i

nh

ibit

ion

Internalization

0.000

10.0

01 0.01 0.1 1 10 10

010

00

1000

0

0

100

200

300

400

500

600CCL3

CCL23

Agonist

% A

ctiv

atio

n

CCR1Efficacy Bias

Arrestin

0.00

001

0.00

010.

001

0.01 0.

1 1 10 100

1000

1000

0

1000

00

0

25

50

75

100

125

150

Lys-BradykininBradykinin[Phe8"Psi"-BDK

Agonist

% A

ctiv

atio

n

Internalization

0.00

001

0.00

010.

001

0.01 0.

1 1 10 100

1000

1000

0

1000

00

0

25

50

75

100

125

Lys-BradykininBradykinin

[Phe8"Psi"-BDK

Agonist

% A

ctiv

atio

n

Calcium

0.00

001

0.00

010.

001

0.01 0.

1 1 10 100

1000

1000

0

1000

00

0

25

50

75

100

125

Lys-BradykininBradykinin[Phe8"Psi"BDK

Agonist

% A

ctiv

atio

n

Potency BiasBDK2

Page 24: GPCR Ligand Bias Assays

Uncover Biased Ligands Using the Complete DiscoveRx GPCR Portfolio

• Opioid system controls responses to pain - OPRD1 (hDOR) activation alleviates persistent pain - Desensitization leads to persistent pain• Receptor responds to endogenous & synthetic ligands• hDOR undergoes rapid internalization• Compound-specific differences in re-sensitization have been identified

Delta Opioid Receptor Case Study

Prolonged G-protein activation

ProlongedDesensitizationFunctional Antagonism

Biased Signaling

2nd Messenger Arrestin Internalization

Page 25: GPCR Ligand Bias Assays

SNC-80 Results in OPRD1 Desensitization

• SNC-80 showed 95% inhibition sustained for 30 minutes

• Faster & stronger desensitization vs endogenous enkephalins

• Fresh ligand could not restore signal

slower

Page 26: GPCR Ligand Bias Assays

•SNC-80 identified as a strongly internalizing compound

•Agonist recycling is compound-specific

SNC-80 Results in Prolonged OPRD1 Internalization

Page 27: GPCR Ligand Bias Assays

In vivo Effects of a Non-Internalizing Ligand

First Challenge

2nd Challeng

eARMS390 and SNC80 are full agonists by GTPgS

SNC80 –EC50: 122nMARMS390-EC50: 170nMSummary• SNC80 is a

strongly internalizing ligand

• SNC80 behaves as a functional antagonist in vivo

Page 28: GPCR Ligand Bias Assays

10-1410-1310 -1210-1110-10 10-9 10-8 10-7 10-6 10-50

5000

10000

15000

20000

25000

Agonist [M] + 20 M Forskolin

RL

U

2nd Messenger

10-1210-1110-10 10-9 10-8 10-7 10-6 10-5 10-4 10-30

50000

100000

150000

200000

Agonist [M]

RL

U

Arrestin

10-1210-1110 -10 10-9 10-8 10-7 10-6 10-5 10-4 10-30

500

1000

1500

2000

Agonist [M]

RL

U

Internalization

DAMGO is a weak agonist compared to SNC-80

DAMGO weakly recruits Arrestin

SNC-80 strong Arrestin response

SNC-80 strong Arrestin-mediated internalization vs DAMGO

Differential GPCR Biology: OPRD1

Page 29: GPCR Ligand Bias Assays

More Information - CCKAR

• Full agonist in calcium

• Similar in vivo half lives

• Why is a compound that is less potent, more active in vivo?

Page 30: GPCR Ligand Bias Assays

Relevant, Predictive, Informative

Arrestin

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

100000

200000

300000

400000sCCK8A-71623

[M]

RL

U

Endo

10-12 10-11 10-10 10-9 10 -8 10-7 10 -6 10-50

1000

2000

3000

4000

5000

6000

7000sCCK8A-71623

[M]

RLU

Working hypothesis: A-71623 has prolonged duration of action due to poor internalization.

2nd Messenger Arrestin Internalization

Potency Efficacy Potency Efficacy Potency Efficacy

Compound X 10nM Full 5nM Full 5nM Partial

Compound Y 50nM Full 35nM Partial 35nM Full

Compound Z 20nM Partial 100nM Full 100nM Full

Moving the industry…….

High Content Compound Analysis

Page 31: GPCR Ligand Bias Assays

Arrestin CHRM2

10-10.0 10-7.5 10-5.0 10-2.5 100.00

50000

100000

150000

[M]

RL

U

Internalization CHRM2

10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-20

2500

5000

7500

10000

12500

[M]

RL

U

cAMP CHRM2

10-10 10 -9 10 -8 10 -7 10-6 10-5 10-40

2500

5000

7500

10000

12500

[M]

RL

U

CHRM2

Uncover Biased Ligands Using the DiscoveRx Complete GPCR Offering: CHRM2

Oxo-M gives a functional response

Behaves as an agonist

Oxo-M shows weak b-Arrestin

recruitment

Oxo-M shows strong Arrestin-mediated internalization

Page 32: GPCR Ligand Bias Assays

Kinetensin behaves as a weak agonist

Kinetensin is a weak agonist, similar EC50s

Kinetensin results in weak internalization

Calcium NTSR1

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

50

100

150

200

250

300

350

[M]

RL

U

Arrestin NTSR1

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

250000

500000

750000

1000000

1250000

[M]

RL

U

Internalization NTSR1

10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40

10000

20000

30000

40000

50000

60000

70000

[M]

RL

U

NTSR1

*Currently building out data sets and algorithms (in collaboration) for Ligand Bias quantification*

Uncover Biased Ligands Using the DiscoveRx Complete GPCR Offering: NTSR1

Page 33: GPCR Ligand Bias Assays

Why Are PathHunter Internalization Assays Such An Important Drug Discovery Tool?• Receptor Internalization Influences Agonist Efficacy- Fewer receptors at the cell surface, reduces potency- Selective and non-selective agonists can be compared

• New classes of drugs can be developed - Functional Antagonists that remove receptors from cell

surface can reduce unwanted side effects-Non-recycling receptors may be better targets for chronic diseases (multiple administrations)

• Molecular Pharmacology - Determine a direct link between

internalization/localization & function for a target

Page 34: GPCR Ligand Bias Assays

DiscoveRx GPCR Portfolio Summary

Multi-Mode GPCR characterization provides: Extensive ligand analysis – identification of biased

ligands Potential to select ligands with specific properties and

durations Correlate biological function with biochemical

characterization Similar Studies

• OPRM1 - μ- Opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization: McPherson et al. Mol Pharm July 2010

• EP4 - Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands: Leduc et al. JPET July 2009

Prolonged G-protein activation

ProlongedDesensitizationFunctional Antagonism

Biased Signaling

2nd Messenger Arrestin Internalization

Page 35: GPCR Ligand Bias Assays

Biased Ligand Discovery with DiscoveRx

Monitor GPCR activity through multiple 7TMR signaling pathways and uncover novel, biased ligands

• Largest GPCR menu covering all 7TMR signaling pathways -- 2nd messenger, arrestin & internalization)

• Preferred supplier offering all technology platforms in the same robust, easy-to-use, HTS-friendly format

• Provide “Next Generation” GPCR platforms that can be used explore novel receptor biology including internalization and dimerization

Page 36: GPCR Ligand Bias Assays

DiscoveRx Comprehensive GPCR Portfolio

GPCR Pathway

DRX Products Advantages

Gi/Gs Native Gi/Gs cell lines,HitHunter™ cAMP XS+

Native, non-force coupled cell lines with chemiluminescent detection

Gq Native Gq cell lines,HitHunter™ Calcium No Wash PLUS

Native, non-force coupled cell lines with fluorescent detection

Gq HitHunter™ IP3 Secondary confirmation of Calcium response

G-protein Independent Signaling

PathHunter™ Arrestin De-orphanization, uncover novel pharmacology biased ligand discovery

PathHunter™ Activated GPCR Internalization Assays

Monitor activated GPCR internalization without imaging, biased ligand discovery

PathHunter™ GPCR Dimerization Assays

Specific and quantitative measure of GPCR heterodimers

Thank You!